A Label-Free Method Combining Chromatography Enrichment with N-Terminal Specific Proteolysis Enables Sensitive Identification of Tn/sTn Glycosites

Tn (GalNAcα1-O-S/T) and sTn (Neu5Acα2-6GalNAcα1-O-S/T) antigens, the immature O-glycans, are strongly correlated with cancer aggressiveness. Tn and sTn antigens exhibit significant differences between patients and healthy people, and they have emerged as potential biomarkers for cancer diagnosis and prognosis. Even though several chemical or chemoenzymatic labeling methods have been developed, the simultaneous analysis of Tn and sTn glycosites at the proteome level still faces great challenges. Here, we propose a label-free method for the sensitive identification of Tn/sTn glycosites from complex proteome samples. Because Tn/sTn antigens often densely occur on protein regions rich in Ser/Thr residues but lacking Lys/Arg residues, the trypsin digestion of these O-glycoproteins will generate relatively large peptides carrying multiple O-glycans. In this study, large tryptic O-glycopeptides were specifically enriched using hydrophilic interaction liquid chromatography. Subsequently, glycopeptides containing a single Tn/sTn antigen were generated by N-terminal-specific protease digestion, enabling the identification of Tn/sTn glycosites at the peptide N-termini. We demonstrated the general applicability of this method to analyze challenging samples, including HeLa cells and pancreatic ductal adenocarcinoma serum, where Tn/sTn are low-abundance O-glycans. Furthermore, a total of 191 Tn/sTn glycosites were identified on 200 glycopeptides from 136 proteins across two pancreatic cancer cell lines and serum. In summary, the method is broadly applicable, sensitive and well fitted to analyze a large cohort of clinical samples.